Full-length single-molecule protein fingerprinting

Mike Filius, Raman van Wee, Carlos de Lannoy, Ilja Westerlaken, Zeshi Li, Sung Hyun Kim, Cecilia de Agrela Pinto, Yunfei Wu, Geert Jan Boons, Martin Pabst, Dick de Ridder, Chirlmin Joo

Research output: Contribution to journalArticlepeer-review

4 Scopus citations

Abstract

Proteins are the primary functional actors of the cell. While proteoform diversity is known to be highly biologically relevant, current protein analysis methods are of limited use for distinguishing proteoforms. Mass spectrometric methods, in particular, often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate fluorescence resonance energy transfer (FRET)-based single-molecule protein fingerprinting to map the location of individual amino acids and post-translational modifications within single full-length protein molecules. Our data show that both intrinsically disordered proteins and folded globular proteins can be fingerprinted with a subnanometer resolution, achieved by probing the amino acids one by one using single-molecule FRET via DNA exchange. This capability was demonstrated through the analysis of alpha-synuclein, an intrinsically disordered protein, by accurately quantifying isoforms in mixtures using a machine learning classifier, and by determining the locations of two O-GlcNAc moieties. Furthermore, we demonstrate fingerprinting of the globular proteins Bcl-2-like protein 1, procalcitonin and S100A9. We anticipate that our ability to perform proteoform identification with the ultimate sensitivity may unlock exciting new venues in proteomics research and biomarker-based diagnosis.

Original languageEnglish
Pages (from-to)652-659
Number of pages8
JournalNature Nanotechnology
Volume19
Issue number5
DOIs
StatePublished - May 2024

Bibliographical note

Publisher Copyright:
© The Author(s), under exclusive licence to Springer Nature Limited 2024.

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